Method for improving pancreatic functions

ABSTRACT

Compounds such as 4-methylcholest-7-en-3-ol, 4-methylergost-7-en-3-ol and 4-methylstigmast-7-en-3-ol which have an activity for improving pancreatic function are used as an active ingredient of a pancreas function improving drug and food or drink.

RELATED APPLICATIONS

This application is the U.S. National Phase under 35 U.S.C. §371 ofInternational Application PCT/JP2006/303709, filed Feb. 28, 2006, whichwas published in a non-English language, which claims priority toJapanese Patent Application No. 2005-144385, filed May 17, 2005.

TECHNICAL FIELD

The present invention relates to a drug and food or drink for improvingpancreatic functions, which contains a compound that can be safelyingested and has an effect for protecting pancreatic endocrine glandcells or improving functions of the pancreatic endocrine gland cells.

BACKGROUND ART

The pancreas is an organ constituted by endocrine gland tissues calledthe pancreas islets (Langerhans islets) and exocrine gland tissuessecreting digestive enzymes. The β cells, α cells, δ cells, pancreaticpolypeptide cells, and so forth exist in the Langerhans islets, and theygreatly affect the control of blood glucose and metabolism. Among these,the β cells play a particularly important role as cells producinginsulin.

Diabetes mellitus is a highly frequently observed metabolic disorderrecognized in 10% of Japanese adults. According to the epidemiology ofthe β cell dysfunction of the pancreas, which is considered one of thecauses of diabetes mellitus, while the β cell dysfunction is of courseobserved in individuals with borderline type hyperglycemia, individualsexhibiting normal glucose tolerance also include individuals exhibitingclearly reduced β cell functions at a rate of 30%. Moreover, it is saidthat adults who lead average social life in present-day Japan highlyfrequently causes insulin resistance more or less, and it is consideredthat, as for persons suffering from insulin resistance, in those who donot suffer from β cell dysfunction, the blood glucose level does notincrease, and in those who suffer from β cell dysfunction, the bloodglucose level increases from a level corresponding to normal glucosetolerance to a level corresponding to borderline type hyperglycemia(Non-patent document 1).

At present, although therapies for promoting spontaneous recovery of thepancreatic functions based on removal of causative pathologicalconditions or factors are used for pancreatic function disorder, anytherapeutic method or agent for positively restoring pancreaticfunctions once degraded has not been used so far, and agents forprotecting pancreatic cells or agents for improving damaged pancreaticcells are desired in the clinical field.

The pancreatic function disorder means a pathological condition that theendocrine or exocrine gland functions of the pancreas are lowered orabnormally enhanced.

As the prior art of agents for curing pancreatic function disorder,those containing neurotrophic factors such as BDNF as an activeingredient (Patent document 1), those containing glycerol derivatives asan active ingredient (Patent document 2), pancreatic function improvingagents containing betacellulin proteins or muteins thereof (Patentdocument 3), and so forth. It has been so far considered that BDNF isreleased from the central end of small DRG neuron with othertransmitters at the time of inflammation or nerve damage, and involvedin promotion of pain signal transduction via tyrosine phosphorylation ofthe NMDA receptor on the dorsal horn cells (Non-patent document 2), andthus it is considered to be restricted for actual use.

Further, the glycerol derivatives disclosed in Patent document 2 are thecompounds described in Patent document 4, and are agents havingantiplatelet-activating factor (PAF) activity for therapeutic andprophylactic treatment of DIC, shock, allergy, acute pancreatitis, braintwitch at the time of subarachnoid haemorrhage, and so forth, and theyare also found to have an organopathy preventing, curing and improvingeffect for preventing, curing and improving organopathy caused inprocesses of preservation of organ in ischemic condition, blood flowobstruction caused by post-graft blood reperfusion or surgery, and soforth. However, it is hard to say that these agents are suitable forchronic pancreatic diseases without these symptoms.

Moreover, the pancreatic function improving agents containingbetacellulin proteins or muteins thereof disclosed in Patent document 3also have an action of acting on undifferentiated pancreatic stem cellsand thereby promoting differentiation of them into the pancreatic βcells producing insulin, and an action of inducing differentiation ofundifferentiated stem cells into other cells of the pancreas such as Fcells producing pancreatic polypeptides, and the effect cannot beexpected under a condition that immature cells are depleted. Inaddition, although mRNAs of these proteins are detected in variousorgans other than the brain, for example, liver, kidney, pancreas, etc.,the details of the functions thereof are not clarified almost at all,and therefore it cannot be said that they can be immediately used forclinical cases.

As techniques of using a triterpene glycoside as an active ingredient,for example, a diabetes preventing agent containing a glycosideextracted from Gymnema inodorum as an active ingredient (Patent document5), a metabolism improving method and a composition therefor containingcorosolic acid extracted from banaba as an active ingredient (Patentdocument 6), a lipase inhibitor (Patent document 7) and a triterpenederivative having an immunosuppressing activity (Patent document 8) havebeen disclosed.

Furthermore, it has been disclosed that compounds having a lanostaneskeleton or 3,4-secolanostane skeleton have an insulin action enhancingactivity (Patent document 9). The effect of these compounds is toenhance the insulin action in regulation of adipocyte differentiation,and the effect thereof on pancreatic diseases remains unknown.

Furthermore, compounds selected from the group consisting of24-alkylcholesten-3-ones and 24-alkylcholestan-3-ones that have nodouble bond in the basic steroid skeleton, or 5-campestene-3-ones thathave double bond between 5 and 6 position which is different from thecompound of the present invention have been disclosed as hypoglycemicagents (Patent document 10).

Meanwhile, lophenol (one of the stereoisomers of4-methyl-cholest-7-en-3-ol) has been known to be an intermediate of thebiosynthetic pathway starting from squalane in plants (Non-patentdocument 3). As for prior arts of this compound, however, there is onlya reference concerning the biosynthetic system of lophenol (Non-patentdocument 4), and the use of the compound is not known at all.

The genus Aloe in the family Liliaceae is a group of plants includingAloe vera (Aloe barbadensis Miller) and Aloe arborescens (Aloearborescens Miller var. natalensis Berger) and so forth, and they areempirically known to have various efficacies. The prior arts regardingthe use of plants of the genus Aloe include immunomodulatingpolysaccharides (Patent document 11), immunosuppression improving agentscontaining a butanol fraction of an aloe extract or aloin (Patentdocument 12), HSP60 family protein synthesis suppressing agentscontaining aloin derivatives (Patent documents 13 to 15), protein havinglectin activity derived from aloe leaf-skin (Patent document 16) andarts regarding improvement of blood glucose levels (Non-patent document5 to 7, Patent document 17 to 20) and so forth.

-   [Patent document 1] International Publication No. WO 00/62796-   [Patent document 2] Japanese Patent Laid-open No. 07-285866-   [Patent document 3] Japanese Patent Laid-open No. 09-188630-   [Patent document 4] Japanese Patent Laid-open No. 10-045604-   [Patent document 5] Japanese Patent Laid-open No. 05-247086-   [Patent document 6] Japanese Patent Laid-open No. 2002-205949-   [Patent document 7] Japanese Patent Laid-open No. 09-040689-   [Patent document 8] Japanese Patent Laid-open No. 11-511482-   [Patent document 9] Japanese Patent Laid-open No. 10-330266-   [Patent document 10] Japanese Patent Laid-open No. 2003-048837-   [Patent document 11] Japanese Patent Laid-open No. 2001-520019-   [Patent document 12] Japanese Patent Laid-open No. 08-208495-   [Patent document 13] Japanese Patent Laid-open No. 10-120576-   [Patent document 14] Japanese Patent Laid-open No. 10-045604-   [Patent document 15] Japanese Patent Laid-open No. 10-036271-   [Patent document 16] Japanese Patent Laid-open No. 09-059298-   [Patent document 17] Japanese Patent Laid-open No. 60-214741-   [Patent document 18] Japanese Patent Laid-open No. 2003-286185-   [Patent document 19] U.S. Pat. No. 4,598,069-   [Patent document 20] U.S. Patent Application Publication No.    2003/0207818-   [Non-patent document 1] “Insulin Resistance,” Diabetes Mellitus    current library, Bunkodo, Apr. 17, 2004-   [Non-patent document 2] Brain Res Rev, Vol. 40, pp. 240-249, 2002-   [Non-patent document 3] Yamada A., “Experimental Methods of    Biochemistry”, Vol. 24, Experimental Methods for Fat and Lipid    Metabolism, p. 174, Gakkai Shuppan Center, 1989-   [Non-patent document 4] Chem. Pharm. Bull., pp. 624-626, 1993-   [Non-patent document 5] Phytomedicine, Vol. 3, pp. 245-248, 1996-   [Non-patent document 6] Phytotherapy Research, Vol. 15, pp. 157-161,    2001-   [Non-patent document 7] Phytotherapy Research, Vol. 7, pp. 37-42,    1993

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a drug and food ordrink suitable for protecting pancreatic endocrine gland cells orimproving functions of the pancreatic endocrine gland cells, which doesnot contain unfavorable ingredients for a drug and food or drink, from araw material that can be safely ingested from experiential viewpoint forfood and is readily obtained.

The inventors of the present invention assiduously studied in order toachieve the foregoing object. As a result, they found that a compoundhaving a structure similar to that of lophenol contained in a wide rangeof plants could be safely ingested and had an activity for improvingpancreatic functions. The present invention was accomplished on thebasis of the above finding.

That is, the present invention provides a drug for improving pancreaticfunctions, which contains a compound represented by the followinggeneral formula (1) as an active ingredient:

In the formula, R1 represents a straight or branched alkyl group having5 to 16 carbon atoms, which may contain no double bond or 1 or 2 doublebonds and may contain a hydroxyl group and/or a carbonyl group, R2 andR3 each independently represent a hydrogen atom, an alkyl group having 1to 3 carbon atoms or a substituted alkyl group having 1 to 3 carbonatoms, and R4 forms C═O with the carbon atom constituting the ring orrepresents —OH or —OCOCH₃.

According to a preferred embodiment, one of R2 and R3 is a hydrogenatom, the other is methyl group, and R4 is a hydroxyl group.

According to a preferred embodiment of the drug of the presentinvention, the improvement of pancreatic functions is to protectpancreatic endocrine gland cells or to improve pancreatic endocrinegland cell functions.

Further, according to a preferred embodiment, in the aforementioned drugand food or drink, R1 is represented by any one of the followingformulas:—CH₂—CH₂—CH(—CH₂—CH₃)—CH(CH₃)₂—CH₂—CH₂—CH═C(CH₃)₂—CH₂—CH═C(CH₃)—CH(CH₃)₂—CH₂—CH₂—C(═CH—CH₃)—CH(CH₃)₂—CH₂—CH₂—CH(Ra)═C(CH₃)Rb

(wherein Ra and Rb is any of —H, —OH, or —CH₃)—CH₂—CH₂—CH(Rc)—CH(CH₃)Rd

(wherein Rc and Rd is any of —H, —OH, or —CH₃)

Furthermore, according to a preferred embodiment, the aforementionedcompound contained in the aforementioned drug and food or drink isselected from the group consisting of 4-methyl-cholest-7-en-3-ol,4-methyl-ergost-7-en-3-ol and 4-methyl-stigmast-7-en-3-ol.

Further, according to a preferred embodiment, the aforementioned drugcontains 0.001 to 10% by dry mass of the aforementioned compound.

Further, according to a preferred embodiment, the aforementioned food ordrink contains 0.0001 to 1% by dry mass of the aforementioned compound.

The present invention further provides a drug for improving pancreaticfunctions, which contains a composition consisting of an organic solventextract or hot water extract of a plant or a fraction thereof containinga compound represented by the aforementioned general formula (1) andcontaining 0.001 to 10% by dry mass of the aforementioned compound as anactive ingredient, or food or drink for improving pancreatic functions,which contains a composition containing 0.0001 to 1% by dry mass of theaforementioned compound as an active ingredient. According to apreferred embodiment of the drug of the present invention, theimprovement of pancreatic functions is to protect pancreatic endocrinegland cells or to improve pancreatic endocrine gland cell functions.

The aforementioned plant used for the aforementioned drug and food ordrink is preferably a plant of the family Liliaceae, and according to aparticularly preferred embodiment, the aforementioned plant of thefamily Liliaceae is a plant classified into the genus Aloe.

The present invention provides the aforementioned food or drink attachedwith an indication that the food or drink is used for improvingpancreatic functions.

Hereafter, the aforementioned drug and food or drink is also genericallyreferred to as “the drug and food or drink of the present invention.”

The present invention further provides use of a compound represented bythe aforementioned general formula (1) or a composition containing thesame in the production of a drug for improving pancreatic functions.According to a preferred embodiment of the use of the present invention,0.001 to 10% by dry mass of the aforementioned compound is contained.According to a preferred embodiment of the use of the present invention,the improvement of pancreatic functions is to protect pancreaticendocrine gland cells or to improve pancreatic endocrine gland cellfunctions.

The present invention further provides a method for improving pancreaticfunctions, which comprises administering a compound represented by theaforementioned chemical formula (1) or a composition containing the sameto a subject whose pancreatic functions are to be improved. According toa preferred embodiment of the method of the present invention, theimprovement of pancreatic functions is to protect pancreatic endocrinegland cells or to improve pancreatic endocrine gland cell functions.Further, according to a preferred embodiment of the method of thepresent invention, the aforementioned composition contains 0.001 to 10%by dry mass of the aforementioned compound.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereafter, preferred embodiments of the present invention will beexplained in detail. However, the present invention is not limited tothe following preferred embodiments, and the preferred embodiments canbe freely modified within the scope of the present invention.

According to an embodiment, the drug and food or drink of the presentinvention contains a compound represented by the aforementioned generalformula (1) having an activity for improving pancreatic functions,especially protecting pancreatic endocrine gland cells or improvingfunctions of pancreatic endocrine gland cells (hereinafter also bereferred to as “the compound of the present invention”) as an activeingredient. According to another embodiment, the drug and food or drinkof the present invention comprises a composition containing an organicsolvent extract or a hot water extract of a plant or a fraction thereofcontaining the compound of the present invention as an activeingredient.

In the aforementioned general formula (1), it is preferred that R1represents a straight or branched alkyl group having 5 to 16 carbonatoms, which may contain no double bond or 1 or 2 double bonds, R2 andR3 each independently represent a hydrogen atom, an alkyl group having 1to 3 carbon atoms or a substituted alkyl group having 1 to 3 carbonatoms, and R4 forms C═O with the carbon atom constituting the ring orrepresents —OH or —OCOCH₃. As the aforementioned alkyl group having 1 to3 carbon atoms, methyl group, ethyl group and so forth are preferred,and methyl group is particularly preferred.

The aforementioned R1 is preferably any one of the groups represented bythe following formulas:—CH₂—CH₂—CH(—CH₂—CH₃)—CH(CH₃)₂  (i)—CH₂—CH₂—CH═C(CH₃)₂  (ii)—CH₂—CH═C(CH₃)—CH(CH₃)₂  (iii)—CH₂—CH₂—C(═CH—CH₃)—CH(CH₃)₂  (iv)—CH₂—CH₂—CH(Ra)═C(CH₃)Rb  (v)

(wherein Ra and Rb is any of —H, —OH, or —CH₃)—CH₂—CH₂—CH(Rc)—CH(CH₃)Rd  (vi)

(wherein Rc and Rd is any of —H, —OH, or —CH₃)

Further, it is preferred that one of R2 or R3 is a hydrogen atom, andthe other is a methyl group. Further, it is preferred that R4 is ahydroxyl group.

The most preferred compounds as the aforementioned compound are thoserepresented by the following formulas, 4-methyl-cholest-7-en-3-ol(formula (2)), 4-methyl-ergost-7-en-3-ol (formula (3)) and4-methyl-stigmast-7-en-3-ol (formula (4)).

That is, 4-methyl-cholest-7-en-3-ol is a compound represented by theaforementioned general formula (1) wherein one of R2 and R3 is ahydrogen atom, the other is methyl group, R4 is a hydroxyl group, and R1is a group represented by the aforementioned formula (vi) (Rc represents—H, and Rd represents —CH₃). 4-Methyl-ergost-7-en-3-ol is a compoundrepresented by the aforementioned general formula (1) wherein one of R2and R3 is a hydrogen atom, the other is methyl group, R4 is a hydroxylgroup, and R1 is a group represented by the aforementioned formula (vi)(Rc and Rd both represent —CH₃). Further, 4-methyl-stigmast-7-en-3-ol isa compound represented by the aforementioned general formula (1) whereinone of R2 and R3 is a hydrogen atom, the other is methyl group, R4 is ahydroxyl group, and R1 is a group represented by the aforementionedformula (i).

The drug and food or drink of the present invention may contain one typeor two or more arbitrary types of the aforementioned compounds.

It is known that lophenol is contained in plants, and the compound ofthe present invention can be produced according to the known method forproducing lophenol (Yamada A., “Experimental Methods of Biochemistry”,Vol. 24, Experimental Methods for Fat and Lipid Metabolism, p. 174,Gakkai Shuppan Center, 1989). The compound of the present invention canbe obtained by, for example, extracting the compound from a plantcontaining the same using a method such as extraction with an organicsolvent or extraction with hot water and purifying the obtained extract.In the present invention, although the compound of the present inventionmay be purified, a composition such as a plant extract or a fractionthereof may also be used so long as it contains an effective amount ofthe compound.

Examples of the aforementioned plant include a plant belonging to thefamily Liliaceae. Examples of the plant belonging to the familyLiliaceae include plants belonging to the genus Aloe or Allium. Examplesof the plants of the genus Aloe include Aloe vera (Aloe barbadensisMiller), Aloe ferox Miller, Aloe africana Miller, Aloe arborescen Millervar. natalensis Berger, Aloe spicata Baker and so forth. In theproduction of the compound of the present invention or a compositioncontaining the same, although the whole of the aforementioned plant maybe used, it is preferable to use mesophyll (clear gel portion) thereof.Such a plant or a part thereof is disrupted preferably by using ahomogenizer or the like and thereby liquefied, and the compound of thepresent invention or a composition containing the same is extracted fromthe disruption product by using an organic solvent or hot water.Examples of the organic solvent include alcohols such as methanol,ethanol and butanol; esters such as methyl acetate, ethyl acetate,propyl acetate and butyl acetate; ketones such as acetone and methylisobutyl ketone; ethers such as diethyl ether and petroleum ether;hydrocarbons such as hexane, cyclohexane, toluene and benzene;halogenated hydrocarbons such as carbon tetrachloride, dichloromethaneand chloroform; heterocyclic compounds such as pyridine; glycols such asethylene glycol; polyhydric alcohols such as polyethylene glycol;nitrile solvents such as acetonitrile, mixtures of these solvents and soforth. Further, these solvents may be anhydrous or hydrous. Among thesesolvents, ethyl acetate/butanol mixture (3:1) and chloroform/methanolmixture (2:1) are particularly preferred.

As the extraction method, a method used for usual extraction of a plantcomponent can be used. Usually used is, for example, a method ofrefluxing 1 to 300 parts by mass of an organic solvent with 1 part bymass of fresh plant or dried plant with heating at a temperature belowthe boiling point of the solvent and stirring or shaking, or a method ofperforming extraction by ultrasonication at room temperature. Byisolating insoluble matters from the extraction liquor using a suitablemethod such as filtration or centrifugation, a crude extract can beobtained.

The crude extract can be purified by various types of chromatographysuch as normal or reverse phase silica gel column chromatography. When agradient of chloroform/methanol mixture is used in normal phase silicagel column chromatography as an elution solvent, the compound of thepresent invention is eluted with a mixing ratio ofchloroform:methanol=about 25:1. Further, when a hexane/ethyl acetatemixture (4:1) is used in reverse phase silica gel column chromatographyas an elution solvent, the compound of the present invention is elutedin a fraction eluted at an early stage.

The obtained fraction can be further purified by HPLC or the like.

Further, the compound used for the present invention may also beproduced by a chemical synthesis method or a biological or enzymaticmethod using microorganisms, enzymes or the like.

Whether a compound or a composition containing the same obtained asdescribed above actually contains the compound of the present inventioncan be confirmed by, for example, mass spectrometry (MS), nuclearmagnetic resonance (NMR) spectroscopy or the like.

The compound of the present invention has a pancreatic functionimproving action, in particular, pancreatic endocrine gland cellprotecting action or pancreatic endocrine gland cell function improvingaction. It can be used as an active ingredient of a drug and food ordrink for improving pancreatic functions, in particular, protectingpancreatic endocrine gland cells or improving pancreatic endocrine glandcell functions. In the present invention, protection of pancreaticendocrine gland cells means to protect the pancreatic endocrine glandcells from denaturation due to various causes, or to prevent decrease ofthe insulin production ability of the pancreatic endocrine gland cells.Further, improvement of pancreatic endocrine gland cell functions meansto enhance the insulin production ability of the pancreatic endocrinegland cells of which insulin production ability decreases. Denaturationof pancreatic endocrine gland cells, or protection of pancreaticendocrine gland cells or improvement of pancreatic endocrine gland cellfunctions can be evaluated by microscopic observation of a pancreatictissue section of an animal or measurement of serum insulin level.

By the aforementioned actions, the compound of the present invention canprevent decrease of the insulin production ability of pancreaticendocrine gland cells, or enhance the insulin production ability ofpancreatic endocrine gland cells of which insulin production abilitydecreases.

As for the db/db mice used in the examples mentioned later, it is knownthat affection of the pancreas is observed in them with aging in termsof week (Science, 153, 1127-1128, 1966). Although it has been reportedthat if N-acetyl-L-cysteine, vitamin C and vitamin E as compounds havingan anti-oxidation action are administered to these mice in combination,decrease of the β cell number in the pancreas can be partially prevented(Diabetes, 48, 2398-2406, 1999), even the dose of onlyN-acetyl-L-cysteine is 100 g/60 kg, and it is expected thatadministration in extremely large doses is required. In contrast,according to the present invention, it can be expected that thepancreatic endocrine gland cell protecting action or the pancreaticendocrine gland cell function improving action can be attained with asmall dose.

The drug of the present invention can be used as an active ingredient ofagents for a prophylactic treatment or therapeutic treatment of diseasescaused by hypofunction of pancreatic endocrine gland cells, for example,pancreatic function disorder in acute pancreatitis, chronicpancreatitis, type I diabetes mellitus, and type II diabetes mellitus,pancreatic hypofunction associating with senile decrease of insulinsecretion, and so forth. Moreover, since the compound of the presentinvention exhibits low toxicity, it can also be used together with anantitumor agent in a treatment of pancreatic cancer. Preferably, anagent used for improving hyperglycemia among the diseases accompanyingdecrease in insulin production ability is not encompassed within thescope of the drug of the present invention.

Furthermore, because leaf-skin of Aloe vera contains barbaloin andaloe-emodin having a laxative action, it is conventionally considered tobe unfavorable as a drug and food or drink for which laxative action isnot expected. Therefore, it is preferred that the composition containingthe compound of the present invention does not contain theseingredients. Further, mesophyll of Aloe vera and a disruption productthereof may also be used as an active ingredient of a drug forprotecting pancreatic endocrine gland cells or improving pancreaticendocrine gland cell functions.

The compound of the present invention can be used as an activeingredient of the drug and food or drink of the present invention as itis. Further, an organic solvent extract or a hot water extract of aplant or a fraction thereof containing the compound of the presentinvention (hereinafter referred to as “extract etc.”) may also be usedas an active ingredient of the drug and food or drink. In this case, theaforementioned extract etc. to be contained in the drug preferablycontains 0.001 to 10% by dry mass, more preferably 0.01 to 1% by drymass, particularly preferably 0.05 to 1% by dry mass, of the compound ofthe present invention. Further, the aforementioned extract etc. to becontained in the food or drink preferably contains 0.0001 to 1% by drymass, more preferably 0.001 to 1% by dry mass, particularly preferably0.005 to 1% by dry mass, of the compound of the present invention. Theaforementioned extract etc. may contain two or more types of thecompound of the present invention. Further, the aforementioned extractetc. may be a solution, or can also be lyophilized or spray-dried in aconventional manner and stored or used as powder.

As the drug of the present invention, the compound of the presentinvention or a composition containing the same per se, or the compoundof the present invention or a composition containing the same combinedwith a pharmaceutically acceptable carrier can be orally or parenterallyadministered to a mammal including human. In the drug of the presentinvention, the compound of the present invention may be apharmaceutically acceptable salt. Examples of the pharmaceuticallyacceptable salt include both metal salts (inorganic salts) and organicsalts including, for example, those listed in “Remington'sPharmaceutical Sciences,” 17th edition, p. 1418, 1985. Specific examplesthereof include, but not limited to, inorganic acid salts such ashydrochloride, sulfate, phosphate, diphosphate, hydrobromate andsulfate, and organic acid salts such as malate, maleate, fumarate,tartarate, succinate, citrate, acetate, lactate, methanesulfonate,p-toluenesulfonate, pamoate, salicylate and stearate. Furthermore, thesalt may be a salt with a metal such as sodium, potassium, calcium,magnesium and aluminum or a salt with an amino acid such as lysine.Furthermore, solvates such as hydrates of the aforementioned compound orpharmaceutically acceptable salts thereof also fall within the scope ofthe present invention.

Dosage form of the drug of the present invention is not particularlylimited and can be suitably selected depending on the therapeuticpurpose. Specific examples thereof include tablet, pill, powder,solution, suspension, emulsion, granules, capsule, syrup, suppository,injection, ointment, patch, eye drop, nasal drop and so forth. For thepreparation, additives generally used in usual therapeutic or preventivedrugs for diseases of internal organs such as pancreas as pharmaceuticalcarriers such as excipients, binders, disintegrating agents, lubricants,stabilizers, flavoring agents, diluents, surfactants and solvents forinjection and so forth can be used. Furthermore, so long as the effectof the present invention is not degraded, the compound of the presentinvention or a composition containing the same can be used incombination with other drugs having pancreatic disease improving orpreventing effect.

Although the amount of the compound of the present invention or acomposition containing the same contained in the drug of the presentinvention is not particularly limited and can be suitably selected, theamount may be, for example, 0.001 to 10% by mass, preferably 0.01 to 1%by mass, particularly preferably 0.05 to 1% by mass, in terms of theamount of the compound of the present invention.

Furthermore, with the drug of the present invention, various diseases,complications and so forth resulted from hypofunction of pancreaticendocrine gland cells can be prevented, and risks of these diseases,complications and so forth can be reduced.

Examples of such various diseases and complications resulted fromhypofunction of pancreatic endocrine gland cells include nerve disorder,nephropathy, retinopathy, cataract, macrovascular disease, diabetesmellitus and so forth.

The administration time of the drug of the present invention is notparticularly limited and can be suitably selected according to themethod for treating an objective disease. Furthermore, theadministration route is preferably determined depending on the dosageform, age, sex and other conditions of patients, severity of symptoms ofpatients and so forth.

The dose of the active ingredient in the drug of the present inventionis suitably selected depending on the dosing regimen, age and sex ofpatients, severity of disease, other conditions of patients and soforth. The amount of the compound of the present invention as an activeingredient is usually selected from the range of, preferably 0.001 to 50mg/kg/day, more preferably 0.01 to 1 mg/kg/day, as a tentative dose.Furthermore, when the composition of the present invention is used, thedry weight of the composition is selected from the range of, preferably0.1 to 1000 mg/kg/day, more preferably 1 to 100 mg/kg/day, as atentative amount. In any case, the dose can be administered once dailyor several times as divided portions.

The compound of the present invention or the extract etc. containing thesame can be added to food or drink. The form and property of the food ordrink are not particularly limited so long as the effect of the activeingredient is not degraded, and the food or drink can be orallyingested, and it can be produced in a conventional manner by using rawmaterials usually used for food or drink except that the aforementionedactive ingredient is added.

The amount of the compound of the present invention or the extract etc.containing the same contained in the food or drink of the presentinvention is not particularly limited and can be suitably selected. Forexample, the compound of the present invention or the extract etc.containing the same is contained in the food or drink in an amount of0.0001 to 1% by mass, preferably 0.001 to 1% by mass, particularlypreferably 0.005 to 1% by mass, in terms of the amount of the compoundof the present invention.

The food or drink of the present invention can be used for variousapplications utilizing the pancreatic endocrine gland cell protectingeffect or pancreatic endocrine gland cell function improving effect. Forexample, it can be used as food or drink suitable for “those who havelow production of insulin”, “those who have low function of insulin”,“those who are getting concerned about their functions of pancreas”,food or drink useful for decreasing or eliminating risk factors oflifestyle-related diseases such as diabetes mellitus caused byhypofunction of pancreas and pancreatitis caused by excessive ingestionof alcohol and stress.

As for the food or drink of the present invention, the expression“protection of pancreatic endocrine gland cells or improvement ofpancreatic endocrine gland cell functions” means that improvement orprevention of various health damages resulted from hypofunction ofpancreatic endocrine gland cells, and “protection of Langerhans isletfunctions”, “improvement of Langerhans islet functions”, “protection ofβ cell functions”, “improvement of β cell functions”, “enhancement ofinsulin production”, “prevention of decrease of insulin production”,“enhancement of insulin activity”, “prevention of decrease of insulinactivity” and so forth are exemplified in the present invention as termshaving a meaning similar to that of the aforementioned “protection ofpancreatic endocrine gland cells or improvement or pancreatic endocrinegland cell functions”.

Furthermore, the food or drink of the present invention is useful for aprophylactic treatment of a disease resulted from hypofunction ofpancreatic endocrine gland cells, for example, pancreatic functiondisorder in acute pancreatitis, chronic pancreatitis, type I diabetesmellitus, and type II diabetes mellitus, hypofunction of pancreasassociating with senile decrease in insulin and so forth. Furthermore,the food or drink of the present invention can be used for aprophylactic treatment of various diseases, complications and so forthresulted from hypofunction of pancreatic endocrine gland cells, and candecrease risks of these diseases, complications and so forth.Furthermore, because the compound of the present invention exhibits lowtoxicity, the food or drink of the present invention is also useful fora patient administered with an antitumor agent in a treatment ofpancreas cancer.

Examples of such various diseases and complications resulted fromhypofunction of pancreatic endocrine gland cells include nerve disorder,nephropathy, retinopathy, cataract, macrovascular disease, diabetes andso forth.

The food or drink of the present invention is preferably marketed asfood or drink attached with an indication that the food or drink is usedfor protection of pancreatic endocrine gland cells or improvement ofpancreatic endocrine gland cell functions, for example, “food or drinkcontaining a compound having a pancreatic endocrine gland cellprotecting effect or a pancreatic endocrine gland cell functionimproving effect indicated as ‘For protection of pancreatic endocrinegland cells or improvement of pancreatic endocrine gland cellfunctions’”, “food or drink containing a plant extract indicated as ‘Forprotection of pancreatic endocrine gland cells or improvement ofpancreatic endocrine gland cell functions’”, “food or drink containingAloe vera extract indicated as ‘For protection of pancreatic endocrinegland cells or improvement of pancreatic endocrine gland cellfunctions’” and so forth.

The wording used for such an indication as mentioned above is notnecessarily be limited to the expression “For protection of pancreaticendocrine gland cells or improvement of pancreatic endocrine gland cellfunctions”, and any other wording expressing the pancreatic endocrinegland cell protecting effect or pancreatic endocrine gland cell functionimproving effect of course falls within the scope of the presentinvention. As such a wording, for example, an indication based onvarious uses allowing consumers to recognize the pancreatic endocrinegland cell protecting effect or pancreatic endocrine gland cell functionimproving effect is also possible. Examples include indications of“Suitable for those who have low production of insulin”, “Suitable forthose who have low function of insulin”, “Useful for decrease orelimination of risk factors (risks) of lifestyle-related diseases suchas diabetes mellitus caused by reduction of insulin activity orproduction, pancreatitis caused by excessive ingestion of alcohol andstress” and so forth.

The aforementioned term “indication” includes all actions for informingconsumers the aforementioned use, and any indications reminding oranalogizing the aforementioned use fall within the scope of the“indication” of the present invention regardless of purpose, content,objective article, medium etc. of the indication. However, theindication is preferably made with an expression that allows consumersto directly recognize the aforementioned use. Specific examples includeactions of indicating the aforementioned use on goods or packages ofgoods relating to the food or drink of the present invention, actions ofassigning, delivering, displaying for the purpose of assigning ordelivering or importing such goods or packages of goods indicated withthe aforementioned use, displaying or distributing advertisements, pricelists or business papers relating the goods with indicating theaforementioned use, or providing information including those as contentswith indicating the aforementioned use by an electromagnetic method(Internet etc.) and so forth.

The indication is preferably an indication approved by theadministration etc. (for example, an indication in a form based on anapproval, which is qualified on the basis of any of various legalsystems provided by the administration), and it is particularlypreferably an indication on advertisement materials at the sales spotssuch as packages, containers, catalogs, pamphlets and POPs, othersdocuments and so forth.

Examples of the indication further include indications as health food,functional food, enteric nutritive food, food for special dietary uses,food with nutrient function claims, quasi-drug and so forth as well asindications approved by the Ministry of Health, Labor and Welfare, forexample, indications approved on the basis of the system of food forspecified health uses and similar systems. Examples of the latterinclude indications as food for specified health uses, indications asfood for specified health uses with qualified health claims, indicationsof influence on body structures and functions, indications of reductionof disease risk claims and so forth, and more precisely, typicalexamples include indications as food for specified health uses(especially indications of use for health) provided in the enforcementregulations of Health Promotion Law (Japan Ministry of Health, Labor andWelfare, Ministerial ordinance No. 86, Apr. 30, 2003) and similarindications.

EXAMPLES

The present invention will be explained more specifically with referenceto the following examples. However, the scope of the present inventionis not limited to the following examples.

Preparation Example

In an amount of 100 kg of mesophyll (clear gel portion) of Aloe vera wasliquefied by using a homogenizer, added with 100 L of an ethylacetate/butanol mixture (3:1) and stirred.

The mixture was left standing overnight, and then the ethylacetate/butanol mixture and the aqueous layer were separated to recoverthe ethyl acetate/butanol mixture. The extract from this ethylacetate/butanol mixture obtained by concentrating the ethylacetate/butanol mixture under reduced pressure weighed 13.5 g. Asolution of 13 g of this extract dissolved in 1 mL of achloroform/methanol mixture (1:1) was loaded on a column filled with 400g of Silica Gel 60 (Merck Ltd.) to attain adsorption of the componentsto the column, then the components were eluted with achloroform/methanol mixture by the stepwise gradient method, in whichthe methanol concentration was increased stepwise (mixing ratios ofchloroform:methanol=100:1, 25:1, 10:1, 5:1 and 1:1), and the eluate wasfractionated for each mixing ratio of the aforementioned mixture. It wasconfirmed by normal phase and reverse phase thin layer chromatography(Merck Ltd., Silica Gel 60F254 and RP-18F2543) that, among thesefractions, the compound of the present invention existed in the fractioneluted with the mixture of chloroform:methanol=25:1.

This crude purified substance (crude purification product 1) containingthe compound of the present invention weighed 3 g. Further, the yieldsof the crude purification products obtained in the above operation fromthe fractions eluted with the mixtures of chloroform:methanol=10:1 and1:1 were 1.17 and 2.27 g, respectively. The solvents of these fractionswere removed, then each extract was dissolved in 1 mL of achloroform/methanol mixture (1:1) and loaded on a column filled with 100g of Silica Gel 60 to attain adsorption of the components to the column,and then the components were eluted with 1100 mL of a hexane/ethylacetate mixture (4:1). The eluted fractions were collected as aliquotsof 300 mL (fraction A), 300 mL (fraction B) and 500 mL (fraction C) inthis order. The yields obtained after removing the solvents from thefractions A, B and C were 0.6 g, 1.35 g and 0.15 g, respectively.

It was confirmed by normal phase and reverse phase thin layerchromatography that the compound of the present invention had beenconcentrated in the fraction A (crude purification product 2). Thiscrude purification product 2 was further separated by HPLC usingCOSMOSIL C18 (Nacalai Tesque, Inc.) with a chloroform/hexane mixture(85:15) to obtain 1.3 mg of compound 3 (4-methyl-cholest-7-en-3-ol), 1.2mg of compound 4 (4-methyl-ergost-7-en-3-ol) and 1 mg of compound 5(4-methyl-stigmast-7-en-3-ol). The structures of these compounds wereconfirmed by MS and NMR.

Test Example 1

In this test, the endocrine gland cell functions (insulin productionability) protecting action of compounds having a lophenol skeleton wasevaluated by using db/db mice known as a model animal of pancreatichypofunction or pancreatic tissue dysfunction.

(1) Preparation of Sample

4-methyl-cholest-7-en-3-ol produced in Preparation Example mentionedabove was used as test sample 1, 4-methyl-ergost-7-en-3-ol was used astest sample 2, and 4-methyl-stigmast-7-en-3-ol was used as test samples3.

(2) Test Method

In this test, 6-week old male db/db mice (purchased from Clea Japan,Inc.) were used. These mice were divided into groups, each consisting of7 animals. Each test sample was dissolved in DMSO, and the concentrationwas adjusted to 0.1 or 1 μg/mL with physiological saline. The final DMSOconcentration was adjusted to 0.2%. The pancreas dysfunction model micewere orally administered with 1 mL of the test sample once a dayeveryday for 42 days with a sonde. The serum insulin level was measuredon the 43rd day of the continuous administration by using Lbis insulinmouse ELISA kit (Shibayagi Co., Ltd).

(3) Test Results

The serum insulin levels on the 43rd day of the continuousadministration of the samples are shown in Table 1. When the test sample1, 2 or 3 was administered at a concentration of 1 μg/animal, the seruminsulin levels were as high as 184, 210 and 211% of that observed in thenegative test respectively, and thus pancreatic function (insulinproduction ability) protecting effect by protecting pancreas dysfunctionwas clearly observed. On the other hand, when it was administered at aconcentration of 0.1 μg/animal, any significant pancreatic functionprotecting effect was not observed. During the administration period, noside-effect was observed at all in view of body weight and pathologicalfindings.

TABLE 1 Serum insulin levels on 43rd day of continuous administrationSerum insulin levels on 43^(rd) day of administration <p relative toSample (ng/mL) negative sample> Negative sample 1.99 ± 0.66 Test sample1 (1 μg) 3.67 ± 0.80 <0.003*> Test sample 1 (0.1 μg) 2.33 ± 1.23 <0.19>Test sample 2 (1 μg) 4.17 ± 2.23 <0.04*> Test sample 2 (0.1 μg) 2.32 ±1.52 <0.27> Test sample 3 (1 μg) 4.20 ± 1.86 <0.02*> Test sample 3 (0.1μg) 2.23 ± 0.68 <0.17> *indicates presence of statistically significantdifference.

Test Example 2

In this test, the pancreatic tissue protecting action of compoundshaving a lophenol skeleton was examined by using db/db mice known as amodel animal of pancreatic hypofunction or pancreatic tissuedysfunction.

(1) Preparation of Sample

4-methyl-cholest-7-en-3-ol produced in Preparation Example mentionedabove was used as test sample 1, 4-methyl-ergost-7-en-3-ol was used astest sample 2, 4-methyl-stigmast-7-en-3-ol was used as test samples 3.Further, β-sitosterol (Tama Biochemical Co., Ltd.) was used as a controlsample.

(2) Test Method

In this test, 6-week old male db/db mice (purchased from Clea Japan,Inc.) were used. These mice were divided into groups, each consisting of7 animals. Each test sample was dissolved in DMSO, and the concentrationwas adjusted to 1 μg/mL with physiological saline. The final DMSOconcentration was adjusted to 0.2%. The model mice were orallyadministered with 1 mL of the test sample once a day everyday for 42days with a sonde. On the 43rd day of the continuous administration, thepancreas was extracted, divided into three portions of upstream,midstream and downstream from the duodenum side, and fixed with aformalin solution, and then paraffin blocks were prepared in aconventional manner. Section slides were prepared from the paraffinblocks, and subjected to hematoxylin-eosin staining. The numbers ofLangerhans islets existing on the sections of the 3 position of thepancreas, and area of Langerhans islet having the maximum area on eachsection were measured by using an ocular micrometer on a microscope(“ECLIPSE E600”, NIKON CORP.).

(3) Test Results

The numbers of Langerhans islets in the pancreatic sections on the 43rdday of the continuous administration of the samples are shown in Table2, and the maximum areas of Langerhans islets on the same day are shownin Table 3. The numbers of Langerhans islets of the mice administeredwith the test sample 1, 2 or 3 were 188, 159 and 150% of the number ofLangerhans islets in the negative sample-administered mice,respectively, and it was found that the numbers were clearly large inthe test sample-administered mice. Similarly, the maximum areas ofLangerhans islets in the mice administered with the test sample 1 or 2maintained 3.6, 4 and 2.8 times as large as that observed in thenegative test, and thus it was found that reduction of the Langerhansislets due to pancreatic dysfunction was prevented. On the other hand,the numbers and maximum areas of Langerhans islets of the miceadministered with the control sample don't show any significant change,and any pancreatic function protecting effect was not observed. Fromthese results, it was revealed that 4-methyl-cholest-7-en-3-ol,4-methyl-ergost-7-en-3-ol, 4-methyl-stigmast-7-en-3-ol had an action ofprotecting pancreatic tissues, especially endocrine cells.

TABLE 2 The numbers of Langerhans islets in pancreatic pathologicsections of treated mice The numbers of Langerhans islets in sections on43^(rd) day of administration <p relative to Sample (piece) negativesample> Negative sample 40.3 ± 9.7  Test sample 1 (1 μg) 75.7 ± 24.7<0.008*> Test sample 2 (1 μg) 64.1 ± 9.1  <0.0003*> Test sample 3 (1 μg)60.4 ± 11.6 <0.004*> Control sample (1 μg) 42.3 ± 20.1 <0.8> *indicatespresence of statistically significant difference.

TABLE 3 Maximum areas of Langerhans islets in pancreatic pathologicsections of treated mice Maximum area of Langerhans islets onadministration × 10³ <p relative to Sample (μm²) negative sample>Negative sample 44.5 ± 17.7 Test sample 1 (1 μg) 160.0 ± 116.0 <0.04*>Test sample 2 (1 μg) 178.7 ± 143.4 <0.005*> Test sample 3 (1 μg) 122.6 ±51.4  <0.006*> Control sample (1 μg) 69.1 ± 40.6 <0.18> *indicatespresence of statistically significant difference.

INDUSTRIAL APPLICABILITY

The drug and food or drink of the present invention can be safelyadministered or ingested and have activity of protecting pancreaticendocrine gland cells and improving pancreatic endocrine gland cellfunctions. Further, the active ingredient of the drug and food or drinkof the present invention can be produced from a plant that can be safelyingested from an experiential viewpoint for food and is readilyavailable, for example, a plant of the family Liliaceae such as Aloevera (Aloe barbadensis Miller).

1. A method for treating pancreatitis, which comprises administering0.001 to 50 mg/kg/day of a compound obtainable from an ethylacetate/butanol mixture extract or a chloroform/methanol mixture extractof a plant of the family Liliaceae or a fraction thereof to a subject inneed of treatment for pancreatitis, wherein the compound is selectedfrom the group consisting of 4-methyl-cholest-7-en-3-ol,4-methyl-ergost-7-en-3-ol and 4-methyl-stigmast-7-en-3-ol.